Methods for treating inflammation pain

ABSTRACT

The present invention relates to methods for treating neurogenic inflammation pain. The methods include administering an effective amount of a composition which includes a botulinum toxin component and a substance P component to a patient, thereby treating the neurogenic inflammation pain.

This application is a divisional and claims priority pursuant to 35U.S.C. § 120 to U.S. patent application Ser. No. 10/082,691, filed Feb.25, 2002, which is hereby incorporated by reference in its entirety.

BACKGROUND

The present invention relates to methods for treating inflammation pain,for example, neurogenic inflammation pain. In particular, the presentinvention relates to methods for treating pain using Clostridial toxincompositions and methods for making the Clostridial toxin compositions.

In recent years attention has been directed toward the interactionbetween components of the nervous system and target cells of the immunesystem. Communication between nerve cells and cells which mediateinflammation is demonstrative of such neuroimmune interactions. Forexample, several studies have demonstrated that mast cells and vascularendothelial cells are often in close contact with nerves and that theremay be a functional interaction between these cells and the nervoussystem. In addition, recent evidence suggests that the neuropeptidesubstance P is an important mediator in cross talk between nerves andinflammation mediating cells.

Mast cells and vascular endothelial cells present a rich source ofinflammation mediating compounds which include histamine,prostaglandins, leukotrienes, neutral proteases, cytokines, bradykininand nitric oxide. It is postulated that in response to stimulus,substance P may be released from nerve cells and then bind toinflammation mediating cells subsequently triggering release of theseand/or other compounds resulting in inflammation. This inflammation isthought to produce pain which includes fibromyalgia pain, migraineheadache, arthritis pain, interstitial cystitis pain, myofascial painsyndrome and irritable bowel pain.

What is needed are methods to stop or reduce the occurrence ofinflammation and inflammation related pain which occurs in response toneural stimulus.

Botulinum Toxin

The anaerobic, gram positive bacterium Clostridium botulinum produces apotent polypeptide neurotoxin, botulinum toxin, which causes aneuroparalytic illness in humans and animals referred to as botulism.The spores of Clostridium botulinum are found in soil and can grow inimproperly sterilized and sealed food containers of home basedcanneries, which are the cause of many of the cases of botulism. Theeffects of botulism typically appear 18 to 36 hours after eating thefoodstuffs infected with a Clostridium botulinum culture or spores. Thebotulinum toxin can apparently pass unattenuated through the lining ofthe gut and attack peripheral motor neurons. Symptoms of botulinum toxinintoxication can progress from difficulty walking, swallowing, andspeaking to paralysis of the respiratory muscles and death.

Botulinum toxin type A (“BoNT/A”) is the most lethal natural biologicalagent known to man. About 50 picograms of botulinum toxin (purifiedneurotoxin complex) serotype A is a LD₅₀ in mice. One unit (U) ofbotulinum toxin is defined as the LD₅₀ upon intraperitoneal injectioninto female Swiss Webster mice weighing 18-20 grams each. Sevenimmunologically distinct botulinum neurotoxins have been characterized,these being respectively botulinum neurotoxin serotypes A, B, C₁, D, E,F and G each of which is distinguished by neutralization withserotype-specific antibodies. The different serotypes of botulinum toxinvary in the animal species that they affect and in the severity andduration of the paralysis they evoke. For example, it has beendetermined that BoNt/A is 500 times more potent, as measured by the rateof paralysis produced in the rat, than is botulinum toxin serotype B(BoNT/B). Additionally, botulinum toxin type B (“BoNt/B”) has beendetermined to be non-toxic in primates at a dose of 480 U/kg which isabout 12 times the primate LD₅₀ for BoNt/A. Botulinum toxin apparentlybinds with high affinity to cholinergic motor neurons, is translocatedinto the neuron and blocks the release of acetylcholine.

Botulinum toxins have been used in clinical settings for the treatmentof neuromuscular disorders characterized by hyperactive skeletalmuscles. Botulinum toxins, and modified botulinum toxins, are alsouseful for the treatment of pain, see for example, U.S. Pat. Nos.6,113,915; 6,333,037; 6,235,289; 5,714,468 and; WO 94/15629 each ofwhich is incorporated in its entirety herein by reference.

BoNt/A has been approved by the U.S. Food and Drug Administration forthe treatment of blepharospasm, strabismus, hemifacial spasm andcervical dystonia. Additionally a botulinum toxin type B has beenapproved by the FDA for the treatment of cervical dystonia. Non-serotypeA botulinum toxin serotypes apparently have a lower potency and/or ashorter duration of activity as compared to BoNt/A. Clinical effects ofperipheral intramuscular BoNt/A are usually seen within one week ofinjection. The typical duration of symptomatic relief from a singleintramuscular injection of BoNt/A averages about three months.

Although all the botulinum toxins serotypes apparently inhibit releaseof the neurotransmitter acetylcholine at the neuromuscular junction,they do so by affecting different neurosecretory proteins and/orcleaving these proteins at different sites. For example, botulinumserotypes A and E both cleave the 25 kiloDalton (kD) synaptosomalassociated protein (SNAP-25), but they target different amino acidsequences within this protein. BoNT/B, D, F and G act onvesicle-associated protein (VAMP, also called synaptobrevin), with eachserotype cleaving the protein at a different site. Finally, botulinumtoxin serotype C₁ (BoNT/C₁) has been shown to cleave both syntaxin andSNAP-25. These differences in mechanism of action may affect therelative potency and/or duration of action of the various botulinumtoxin serotypes.

Regardless of serotype, the molecular mechanism of toxin intoxicationappears to be similar and to involve at least three steps or stages. Inthe first step of the process, the toxin binds to the presynapticmembrane of the target neuron through a specific interaction between theH chain and a cell surface receptor; the receptor is thought to bedifferent for each serotype of botulinum toxin and for tetanus toxin.The carboxyl end segment of the H chain, H_(c), appears to be importantfor targeting of the toxin to the cell surface.

In the second step, the toxin crosses the plasma membrane of thepoisoned cell. The toxin is first engulfed by the cell throughreceptor-mediated endocytosis, and an endosome containing the toxin isformed. The toxin then escapes the endosome into the cytoplasm of thecell. This last step is thought to be mediated by the amino end segmentof the H chain, H_(N), which triggers a conformational change of thetoxin in response to a pH of about 5.5 or lower. Endosomes are known topossess a proton pump which decreases intra endosomal pH. Theconformational shift exposes hydrophobic residues in the toxin, whichpermits the toxin to embed itself in the endosomal membrane. The toxinthen translocates through the endosomal membrane into the cytosol.

The last step of the mechanism of botulinum toxin activity appears toinvolve reduction of the disulfide bond joining the H and L chain. Theentire toxic activity of botulinum and tetanus toxins is contained inthe L chain of the holotoxin; the L chain is a zinc (Zn++) endopeptidasewhich selectively cleaves proteins essential for recognition and dockingof neurotransmitter-containing vesicles with the cytoplasmic surface ofthe plasma membrane, and fusion of the vesicles with the plasmamembrane. Tetanus neurotoxin, botulinum toxin, B, D, F, and G causedegradation of synaptobrevin (also called vesicle-associated membraneprotein (VAMP)), a synaptosomal membrane protein. Most of the VAMPpresent at the cytosolic surface of the synaptic vesicle is removed as aresult of any one of these cleavage events. Each toxin specificallycleaves a different bond.

The molecular weight of the botulinum toxin protein molecule, for allseven of the known botulinum toxin serotypes, is about 150 kD.Interestingly, the botulinum toxins are released by Clostridialbacterium as complexes comprising the 150 kD botulinum toxin proteinmolecule along with associated non-toxin proteins. Thus, the BoNt/Acomplex can be produced by Clostridial bacterium as 900 kD, 500 kD and300 kD forms. BoNT/B and C₁ are apparently produced as only a 500 kDcomplex. BoNT/D is produced as both 300 kD and 500 kD complexes.Finally, BoNT/E and F are produced as only approximately 300 kDcomplexes. The complexes (i.e. molecular weight greater than about 150kD) are believed to contain a non-toxin hemagglutinin protein and anon-toxin and non-toxic nonhemagglutinin protein. These two non-toxinproteins (which along with the botulinum toxin molecule comprise therelevant neurotoxin complex) may act to provide stability againstdenaturation to the botulinum toxin molecule and protection againstdigestive acids when toxin is ingested. Additionally, it is possiblethat the larger (greater than about 150 kD molecular weight) botulinumtoxin complexes may result in a slower rate of diffusion of thebotulinum toxin away from a site of intramuscular injection of abotulinum toxin complex.

In vitro studies have indicated that botulinum toxin inhibits potassiumcation induced release of both acetylcholine and norepinephrine fromprimary cell cultures of brainstem tissue. Additionally, it has beenreported that botulinum toxin inhibits the evoked release of bothglycine and glutamate in primary cultures of spinal cord neurons andthat in brain synaptosome preparations botulinum toxin inhibits therelease of each of the neurotransmitters acetylcholine, dopamine,norepinephrine, CGRP and glutamate.

BoNt/A can be obtained by establishing and growing cultures ofClostridium botulinum in a fermenter and then harvesting and purifyingthe fermented mixture in accordance with known procedures. All thebotulinum toxin serotypes are initially synthesized as inactive singlechain proteins which must be cleaved or nicked by proteases to becomeneuroactive. The bacterial strains that make botulinum toxin serotypes Aand G possess endogenous proteases and serotypes A and G can thereforebe recovered from bacterial cultures in predominantly their active form.In contrast, botulinum toxin serotypes C₁, D and E are synthesized bynonproteolytic strains and are therefore typically unactivated whenrecovered from culture. Serotypes B and F are produced by bothproteolytic and nonproteolytic strains and therefore can be recovered ineither the active or inactive form. However, even the proteolyticstrains that produce, for example, the BoNt/B serotype only cleave aportion of the toxin produced. The exact proportion of nicked tounnicked molecules depends on the length of incubation and thetemperature of the culture. Therefore, a certain percentage of anypreparation of, for example, the BoNt/B toxin is likely to be inactive,possibly accounting for the known significantly lower potency of BoNt/Bas compared to BoNt/A. The presence of inactive botulinum toxinmolecules in a clinical preparation will contribute to the overallprotein load of the preparation, which has been linked to increasedantigenicity, without contributing to its clinical efficacy.Additionally, it is known that BoNt/B has, upon intramuscular injection,a shorter duration of activity and is also less potent than BoNt/A atthe same dose level.

It has been reported (as exemplary examples) that BoNt/A has been usedclinically as follows:

(1) about 75-125 units of BOTOX®¹ per intramuscular injection (multiplemuscles) to treat cervical dystonia;¹Available from Allergan, Inc., of Irvine, Calif. under the tradenameBOTOX®.

(2) 5-10 units of BOTOX® per intramuscular injection to treat glabellarlines (brow furrows) (5 units injected intramuscularly into the procerusmuscle and 10 units injected intramuscularly into each corrugatorsupercilii muscle);

(3) about 30-80 units of BOTOX® to treat constipation by intrasphincterinjection of the puborectalis muscle;

(4) about 1-5 units per muscle of intramuscularly injected BOTOX® totreat blepharospasm by injecting the lateral pre-tarsal orbicularisoculi muscle of the upper lid and the lateral pre-tarsal orbicularisoculi of the lower lid.

(5) to treat strabismus, extraocular muscles have been injectedintramuscularly with between about 1-5 units of BOTOX®, the amountinjected varying based upon both the size of the muscle to be injectedand the extent of muscle paralysis desired (i.e. amount of dioptercorrection desired).

(6) to treat upper limb spasticity following stroke by intramuscularinjections of BOTOX® into five different upper limb flexor muscles, asfollows:

-   -   (a) flexor digitorum profundus: 7.5 U to 30 U    -   (b) flexor digitorum sublimus: 7.5 U to 30 U    -   (c) flexor carpi ulnaris: 10 U to 40 U    -   (d) flexor carpi radialis: 15 U to 60 U    -   (e) biceps brachii: 50 U to 200 U. Each of the five indicated        muscles has been injected at the same treatment session, so that        the patient receives from 90 U to 360 U of upper limb flexor        muscle BOTOX® by intramuscular injection at each treatment        session.

The tetanus neurotoxin acts mainly in the central nervous system, whilebotulinum neurotoxin acts at the neuromuscular junction; both act byinhibiting acetylcholine release from the axon of the affected neuroninto the synapse, resulting in paralysis. The effect of intoxication onthe affected neuron is long lasting and until recently has been thoughtto be irreversible. The tetanus neurotoxin is known to exist in oneimmunologically distinct serotype.

SUMMARY

The present invention provides for methods to treat various types ofinflammation and pain associated with inflammation.

In accordance with the present invention there are provided methods fortreating neurogenic inflammation and neurogenic inflammation pain. Themethods may include administering an effective amount of a compositionwhich includes a botulinum toxin component and a substance P componentto a patient thereby treating the neurogenic inflammation and/orneurogenic inflammation pain.

The botulinum toxin component may include an H_(N) and an L chain. Forexample, the H_(N) may be obtained from a botulinum toxin serotype A,serotype B, serotype C, serotype D, serotype E, serotype F or serotypeG. Also for example, the L chain may be obtained from a botulinum toxinserotype A, serotype B, serotype C, serotype D, serotype E, serotype For serotype G.

In accordance with the invention the substance P component may be asubstance P, a precursor of substance P or a substance P analogue.

The present invention provides for methods for treating pain caused byinflammation including fibromyalgia pain, myofascial pain syndrome pain,arthritis pain, migraine headache pain, irritable bowel syndrome pain,Crohn's disease pain and interstitial cystitis pain.

In accordance with the present invention, the compositions may beadministered subcutaneously, intramuscularly or systemically. Thecompositions may be administered with a needle, by needleless injectionorally, by inhalable delivery methods such as an inhalable mist or likemethods.

Methods of the present invention provide for pain relief ranging fromabout 5% to about 100% in effectiveness. For example, pain may bereduced by about 20%, about 40%, about 50%, about 60%, about 80% orabout 100%.

The present invention also provides for methods for inhibiting paincaused by degranulation of mast cells and/or release of inflammationmediating compounds from vascular endothelial cells. The method mayinclude administering to a patient an effective amount of a compositionwhich includes a botulinum toxin component attached to a substance Pcomponent, thereby inhibiting pain caused by degranulation of mast cellsand/or release of inflammation mediating compounds from vascularendothelial cells. For example, the present invention provides formethods for inhibiting pain caused by histamine release from mast cells.These methods may include administering to a patient an effective amountof a composition which includes a botulinum toxin component attached toa substance P component, thereby inhibiting pain caused by histaminerelease from mast cells.

Each and every feature described herein, and each and every combinationof two or more of such features, is included within the scope of thepresent invention provided that the features included in such acombination are not mutually inconsistent.

Definitions

An “agent” is defined as a modified neurotoxin that possesses some orall of the biological activity biological activity of an unmodifiedneurotoxin. Modified neurotoxins include variants and fragments ofneurotoxins. One example of a modified neurotoxin, as disclosed herein,is a portion of a botulinum toxin coupled to a substance P molecule.

A “clostridial neurotoxin” may refer to an intact toxin for example abotulinum toxin, butyricum toxin or tetani toxin or a fragment orportion of a toxin for example a fragment or portion of a botulimumtoxin, butyricum toxin or tetani toxin.

“H_(C)” means a fragment obtained from the H chain of a Clostridialtoxin which is equivalent, for example functionally equivalent, to thecarboxyl end fragment of the H chain, or the portion corresponding tothat fragment in the intact H chain involved in binding to a cellsurface or cell surface receptor.

“H_(N)” means a fragment or variant obtained from an H chain of aClostridial toxin which may be functionally equivalent to the portion ofan intact H chain involved in the translocation of at least the L chainacross an intracellular endosomal membrane into a cytoplasm of a cell.An H_(N), may result from an H_(C) being removed from an H chain. AnH_(N) may also result from an H chain being modified such that its H_(C)no longer binds to cholinergic cell surfaces.

“Heavy chain” means the heavy chain of a clostridial neurotoxin or afragment or variant of an H_(N) of a clostridial neurotoxin. A heavychain may have a molecular weight of about 100 kDa and can be referredto as H chain, or as H.

“LH_(N)” means a fragment obtained from a clostridial neurotoxin thatcontains the L chain coupled to an H_(N). LH_(N) can be obtained fromthe intact clostridial neurotoxin by proteolysis, so as to remove or tomodify the H_(C) domain.

“Light chain” means the light chain of a clostridial neurotoxin or afragment or variant of a light chain of a clostridial neurotoxin. Alight chain may have a molecular weight of about 50 kDa, and can bereferred to as L chain, L, or as the proteolytic domain (amino acidsequence) of a clostridial neurotoxin.

“Linker” means a molecule which couples two or more other molecules orcomponents together.

“Local administration” means direct administration by a non-systemicroute at or in the vicinity of the site of an affliction, disorder, orperceived pain.

“Neurogenic” means arising from the nervous system. For example,neurogenic inflammation refers to inflammation that may be caused, atleast in part, directly or indirectly, by the nervous system.

“Spacer” means a molecule or set of molecules which physically separateand add distance between the components. One function of a spacer may beto prevent steric hindrance between the components.

“Substantial or Substantially” means largely but not entirely. Forexample, substantial relief of pain may mean pain relief of 10%, 20%,30%, 40% 50% or more.

“Targeting moiety” means a molecule that has a specific binding affinityfor a cell surface or cell surface receptor.

“Variable region” means the part of an antibody that varies extensivelyfrom one antibody to another as a result of alternative subunitsequences.

“Variant” means a molecule or peptide which is substantially the same asthat of a disclosed molecule or peptide in its structure and function.For example, a variant of a specified light chain may havenon-consequential amino acid sequence substitutions when compared to theamino acid sequence of the specified light chain. Variants may beconsidered to be equivalent to the specifically disclosed molecules andas such are within the scope of the invention.

DESCRIPTION

This invention is based upon the discovery that inflammation pain can betreated by administering to a patient an agent which includes aclostridial neurotoxin component and a targeting moiety component,wherein the targeting moiety component may bind to cells involved inmediating inflammation.

The mechanism of action for these agents in alleviating pain iscurrently not fully understood. However, without wishing to limit theinvention to any particular theory or mechanism of operation, it isbelieved that the agents disclosed herein may target cells, for example,mast cells and/or vascular endothelial cells which may have receptorsfor the targeting moieties, for example, a substance P targeting moiety.

Mast cells and vascular endothelial cells present many distinctbiologically active, inflammation mediators that may include histamine,prostaglandins, leukotrienes, neutral proteases, cytokines bradykininand nitric oxide. Release of these and/or other mediators may contributeto the events which cause neurogenic inflammation. Further examples ofmediators that may contribute to neurogenic inflammation are discussedin First (U.S. Pat. No. 6,063,768) which is incorporated in its entiretyherein by reference.

Sensory neurons containing substance P are thought to be involved inregulating the inflammatory and immune response in the peripheraltissues that they innervate. It is theorized that these sensory neuronsrelease substance P which binds to and causes secretion frominflammation mediating cells. For example, substance P may bind to mastcell receptor sites thereafter triggering degranulation which includessecretion from mast cells of histamine, prostiglandins, leukotrines,serotonin and other molecules that may serve as inflammatory mediators(Spanos et al. J of Urology Vol 157 p 669-672; Toyoda et al ArchDermatol Res Vol 292 p 418-421). In another example, substance P maytrigger release of inflammation mediators such as bradykinin, nitricoxide and vasoactive intestinal peptide from vascular endothelial cells.

It is theorized that inflammatory mediator release triggered bysubstance P begins with the initial binding of substance P to specificreceptors which may be mobile and randomly distributed on the membranesurface of certain cells, for example, mast cells and/or vascularendothelial cells. These substance P-receptor complexes may accumulatein specialized regions of the membrane termed coated pits. From thisstage, receptor mediated endocytosis (RME) may proceed to the formationof smooth-walled vesicles which allow entry of the substance P-receptorcomplexes into the cell. These vesicles, often referred to as“endosomes” or “receptosomes,” may fuse together or combine to producelarger vesicles. Subsequently, the internal pH of the endosomes may bedecreased by the action of proton pumps. This decrease in pH may changethe conformation of the receptor and/or substance P resulting in therelease of the substance P from the receptor and the formation ofseparate receptor-containing vesicles and substance P-containingcontaining vesicles. The resulting substance P vesicles along with theinternalized substance P, may be delivered to and fused with lysosomeswhere the eventual breakdown of the vesicles and release of thesubstance P likely takes place.

It is thought that at some point after formation of the substanceP-receptor complex, possibly after internalization of the complex,release of inflammation mediators is triggered.

During secretion or exocytosis, the mediators may be included invesicles which fuse to the inner surface of the cell membrane therebyreleasing the vesicle contents to the outside of the cell. Withoutwishing to limit the present invention to any theory or mechanism ofoperation, it is theorized that interference with the exocytosis processmay be the mode of action of the agents of the present invention.

It is theorized that agents of the present invention may operate byfirst targeting inflammation mediating cells by use of a targetingmoiety component such as substance P, and then prevent or reduce thesecretion of inflammation producing molecules by cleaving or byotherwise interfering with the function of proteins involved in thesecretory process by use of a light chain component, for example, abotulinum light chain component. A heavy chain component, for exampleH_(N), may also function in certain embodiments of the present inventionby, for example, assisting in the release of an agent of the inventionfrom intracellular vesicles, for example, endosomes.

The agents described for use in this invention may be very specific fortreating inflammation pain without being cytotoxic because they may notsubstantially or significantly interact with and/or interfere withneurons. Also, the agents can alleviate pain without being cytotoxic totheir target neurons. Because of lack of neural interaction and lack ofcytotoxicity, agents within the scope of the present invention can beadministered either locally at or near sites of inflammation orinflammation pain or systemically.

Agents may be used to treat conditions cause by inflammation, forexample neurogenic inflammation. Examples of conditions caused byneurogenic inflammation that may be treated by methods of the presentinvention include pain associated with fibromyalgia, certain forms ofarthritis, myofascial pain syndrome, interstitial cystitis and irritablebowel syndrome. Additional examples of conditions that may be treated inaccordance with the present invention include myasthenia gravis,systemic lupus erythematosus, discoid lupus erythematosus, organtransplant, tissue transplant, fluid transplant, Graves disease,thyrotoxicosis, autoimmune diabetes, haemolytic anaemia,thrombocytopenic purpura, neutropenia, chronic autoimmune hepatitis,autoimmune gastritis, pernicious anaemia, Hashimoto's thyroiditis,Addison's disease, Sjogren's syndrome, primary biliar cirrhosis,polymyositis, scleroderma, systemic sclerosis, pemphigus vulgaris,bullous pemphigoid, myocarditis, rheumatic carditis, glomerulonephritis,uveitis, orchitis, ulcerative colitis, vasculitis, atrophic gastritis,pernicious anaemia, type 1 diabetes mellitus. Further examples ofconditions that may be caused by neurogenic inflammation that may betreated by methods of the present invention are discussed in First (U.S.Pat. No. 6,063,768).

According to one broad aspect of the invention, methods are provided forthe use of a clostridial neurotoxin component covalently coupled to atargeting moiety. The clostridial neurotoxin component may be obtainedfrom Clostridium beratti, Clostridium butyricum, or Clostridiumbotulinum, for example, Clostridium botulinum toxin types A, B, C, D, E,F and G may be used.

The clostridial neurotoxin component may include only a fragment of theentire neurotoxin. For example, it is known in the art that the H_(C) ofthe neurotoxin molecule can be removed from the other segment of the Hchain, the H_(N), such that the H_(N) fragment remains disulphide linkedto the L chain of the neurotoxin molecule to provide a fragment known asthe LH_(N). In addition, LH_(N) can be produced by modifying the H_(C)fragment to reduce or eliminate the targeting affinity or ability ofH_(C) to bind to its native target site. H_(C) can be modified bymethods known in the art, for example, alterations to the amino acidsequence of the H_(C) or chemical modification to the H_(C).Furthermore, the light chain or a fragment of the light chain, with noassociated H chain or H chain fragment, can be coupled to a targetingmoiety in accordance with the present invention. Therefore, in oneembodiment of the present invention LH_(N) of a clostridial neurotoxinis covalently coupled to a targeting moiety, for example, substance P.In another embodiment, the L chain of a clostridial neurotoxin iscovalently coupled to a targeting moiety, for example, substance P.

In another embodiment, the agent includes an H chain of a clostridialneurotoxin, in which the H_(C) is removed, mutated or chemicallymodified to reduce or eliminate the ability of H_(C) to bind to theneurotoxin receptors at the neuromuscular junction combined with theL-chain of a different clostridial neurotoxin, to form a hybrid. Forexample, in one embodiment, the clostridial neurotoxin componentcomprises an H chain with the H_(C) removed, mutated or chemicallymodified obtained from botulinum toxin type A, and an L chain obtainedfrom another botulinum toxin type. The described hybrid is covalentlycoupled to a targeting moiety.

In another embodiment, the agent includes an L chain of a clostridialneurotoxin, or a fragment of the L chain containing the endopeptidaseactivity, linked to a targeting moiety.

In a broad embodiment, spacers may be used to physically separatecomponents of agents used in accordance with the present invention. Forexample, an agent of the present invention may comprise an L chain or aLH_(N) connected to a targeting moiety, for example substance P, througha spacer. A spacer may function to create a distance between thecomponents to minimize or eliminate steric hindrance of the componentsthat may otherwise occur.

In one embodiment, the spacer region is made up of sugar molecules, forexample, saccharides, glucose, etc. In another embodiment, the spacerregion may be constructed from an aliphatic chain. In anotherembodiment, the spacer regions may be constructed by linking together aseries of amino acids, preferably glycine because glycine are small andare devoid of any functional group. In yet another embodiment, thespacer region may comprise one or more of the sugar molecules, aliphaticchains, and amino acids.

In one embodiment, a spacer comprises a proline, serine, threonineand/or cysteine-rich amino acid sequence similar or identical to animmunoglobulin hinge region. For example, the spacer may comprise theamino acid sequence of a human immunoglobulin g1 hinge region.

Spacers may comprise hydrocarbon moieties, for example, hydrocarbonmoieties represented by the chemical formulas:HOOC—(CH₂)_(n)—COOH, where n=1-12 or,HO—(CH₂)_(n)—COOH, where n>10

In one embodiment, linkers may be used to link together two or moremolecules, components and/or spacers. For example, a Linker may be usedto link a targeting moiety to a L or LH_(N). In another embodiment, aLinker (Y) may be employed to link an L or LH_(N) to a spacer; in turn,that spacer may then be linked to targeting moiety by another Linker(Y), forming, for example, an agent comprising the structure:

L-Y-Spacer-Y-Targeting Moiety.

Examples of substances that can be used as linker (Y) are2-iminothiolane, N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),4-succinimidyloxycarbonyl-alpha-(2-pyridyidithio)toluene (SMPT),m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS),N-succinimidyl(4-iodoacetyl)aminobenzoate (SIAB), succinimidyl4-(p-maleimidophenyl)butyrate (SMPB),1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), bis-diazobenzidineand glutaraldehyde.

In one embodiment, a linker may be attached to an amino group, acarboxylic group, a sulfhydryl group or a hydroxyl group or an aminogroup of a component. For example, a linker may be linked to a carboxylacid group of an amino acid of a targeting moiety.

Although the described chemistry may be used to couple components ofagents used in the invention, any other coupling chemistry known tothose skilled in the art capable of chemically attaching one componentto another component of an agent of the invention is included within thescope of the present invention.

In one embodiment, an agent comprises an H_(N) and/or an L chain and atargeting moiety, covalently linked together. For example, the H_(N) maybe linked to the L chain and the L chain may be linked to the targetingmoiety. In another example, the H_(N) may be linked to the targetingmoiety and to the L chain. In still another example, the targetingmoiety may be linked to H_(N) and the L chain. In still another example,the L chain may be linked to the H_(N) and the targeting moiety. Linkersand/or spacers may be used between some, all or none of the componentsin these examples.

In another embodiment, the targeting moieties may be components that aresubstantially similar to substance P. These components include substanceP precursors, fragments and analogs. The history, isolation,identification, and synthesis of substance P and its precursors,fragments and analogs are disclosed in U.S. Pat. No. 5,891,842(incorporated herein by reference in its entirety).

Substance P is an 11 amino acid peptide which has a number of differentnatural and synthetic precursor forms; has been demonstrated to beconverted into a variety of naturally occurring amino-terminal peptidefragments; and can be obtained in analog format compromising,substituted counterparts thereof, for example, lysine methyl ester,D-amino acids or disulfide bridges substitutions, which may therebyyield more stable and discriminating formulations. A representativelisting of substance P and its related chemical entities is provided byTable I below. The amino acid sequence (1) in Table I(Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-amide) can be referred toas SEQ ID NO: 1, and the subsequent 17 amino acid sequences set forth inTable one can be similarly identified as SEQ ID NO:2 to SEQ ID NO:18.TABLE 1 Substance P, and Representative Precursors, Fragments andStabilized Or Substituted Analogs Name Formula (1) Substance PArg-Pro-Lys-Pro-Gln-Gln-Phe- Phe-Gly-Leu-Met-amide Natural Precursors:(2) Substance P-Glycine* Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-Gly (3) Substance P-Glycine-Lysine*Arg-Pro-Lys-Pro-Gln-Gln-Phe- Phe-Gly-Leu-Met-Gly-Lys (4) SubstanceP-Glycine-Lysine Arg-Pro-Lys-Pro-Gln-Gln-Phe- Arginine*Phe-Gly-Leu-Met-Gly-Lys-Arg Carboxy-EsterSynthetic Precursors: (5)Substance P-Glycine Methyl Arg-Pro-Lys-Pro-Gln-Gln-Phe- Ester°Phe-Gly-Leu-Met-Gly-OMe (6) Substance P-Glycine-LysineArg-Pro-Lys-Pro-Gln-Gln-Phe- Methyl Ester° Phe-Gly-Leu-Met-Gly-Lys-OMe(7) Substance P-Glycine-Lysine Arg-Pro-Lys-Pro-Gln-Gln-Phe- ArginineMethyl Ester° Phe-Gly-Leu-Met-Gly-Lys-Arg-OMe (8) Substance P-GlycineEthyl Arg-Pro-Lys-Pro-Gln-Gln-Phe- Ester° Phe-Gly-Leu-Met-Gly-OEth (9)Substance P-Glycine-Lysine Arg-Pro-Lys-Pro-Gln-Gln-Phe- Ethyl Ester°Phe-Gly-Leu-Met-Gly-Lys-OEth (10)Substance P-Glycine-LysineArg-Pro-Lys-Pro-Gln-Gln-Phe- Arginine Ethyl Ester°Phe-Gly-Leu-Met-Gly-Lys-Arg-OEth Naturally-Occurring Amino-TerminalPeptide Fragments: (11)Substance P/1-4# Arg-Pro-Lys-Pro (12)SubstanceP/1-7# Arg-Pro-Lys-Pro-Gln-Gln-Phe (13)Substance P/1-9#Arg-Pro-Lys-Pro-Gln-Gln-Phe- Phe-Gly Analogs Comprising SyntheticD-Amino Acids Or Disulfide Cys-Cys) Bridges: (14)[D-Pro2, D-Phe7,Arg-D-Pro-Lys-Pro-Gln-Gln-D-Phe- D-Trp9]-Substance P^(¢)Phe-D-Trp-Leu-Met-amide (15)[D-Pro2, D-Phe7,Arg-D-Pro-Lys-Pro-Gln-Gln-D-Phe- D-Trp9]-Substance P-Glycine^(¢))Phe-D-Trp-Leu-Met-Gly (16)[D-Pro2, D-Trp7,Arg-D-Pro-Lys-Pro-Gln-Gln-D-Trp- D-Trp9]-Substance P^(¢)Phe-D-Trp-Leu-Met-amide (17)[D-Pro2, D-Trp7,Arg-D-Pro-Lys-Pro-Gln-Gln-D-Trp- D-Trp9]-Substance P-Glycine^(¢)Phe-D-Trp-Leu-Met-Gly (18)[Cys3, Cys6, Tyr8,Arg-Pro-Cys-Pro-Gln-Cys-Phe-Tyr- Pro10]-Substance P^(¢)Gly-Pro-Met-amide*Shimonka et al., J. Neurochem. 59: 81-92 (1992)°Lee et al., Eur. J. Biochem. 114: 315-327 (1981); Pernow, B.,Pharmacol. Rev. 35: 86-138 (1983); and Regoli et al., TIPS 9: 290-295(1988).#Stewart et al., Nature 262: 784-785 (1986); and Skilling et al., J.Neurosci. 10: 1309-1318 (1990)^(¢)Lavielle et al., Biochem. Pharmacol. 37: 41 (1988); and Quirion, R.and T. V. Dam, Regulatory Peptides 22: 18 (1988)

In one embodiment, agents for use in accordance with the inventioncomprise a hybrid of two clostridial neurotoxins. For example, the Hchain, in one embodiment H_(N), may be obtained from botulinum toxin A,B, C, D, E, F or G and the L chain obtained from another botulinum toxinselected from botulinum toxin A, B, C, D, E, F or G. The two chains fromdifferent botulinum toxin serotypes may be joined together by covalentbonds and/or by disulfide bonds and/or by hydrogen bonds and/or by ionicbonds to for a hybrid LH_(N). This hybrid LH_(N) may be linked to atargeting moiety, for example, substance P or a component that issubstantially similar to substance P, for example, substance Pprecursors, substance P analogs and substance P fragments, by standardmethodologies known in the art. In one embodiment the targeting moietyis covalently linked to a hybrid LH_(N).

In another embodiment, the agents comprises an L chain of a clostridialneurotoxin, or a fragment of an L chain containing the endopeptidaseactivity, coupled to substance P or component that is substantiallysimilar to substance P. The L chain or fragment of the L chain isobtained from botulinum toxin A, B, C, D, E or G and is coupled tosubstance P or components that are substantially similar to substance P,for example, substance P precursors, substance P analogs and substance Pfragments, by standard methodologies known in the art. Linkers and/orspacer components may be used in the coupling.

An agent used in accordance with the present invention may comprise aheavy chain or a portion of a heavy chain of a clostridial neurotoxin.Even more preferably, the H_(N) of the heavy chain is able to facilitatethe transfer of the agent across an endosome membrane into the cytosolof a cell, for example, a mast cell. The clostridial neurotoxin heavychain may be obtained from Clostridium botulinum neurotoxin type A. Inother embodiments, the heavy chain may be obtained from Clostridiumbotulinum types B, C, D, E, F, G and mixtures thereof. Also, the heavychain may be obtained from neurotoxins obtained from Clostridium baratiiand Clostridium butyricum or Clostridium tetani.

Agents that may be used in accordance with the present invention arealso disclosed in U.S. patent application Ser. Nos. 09/489,667,09/922,093 and 09/625,098 which are incorporated in their entiretyherein by reference.

According to another broad aspect of this invention, recombinant DNAmethodologies may be used to produce the components of agents useful inaccordance with the invention, including the targeting moiety, the lightchain or light chain fragment and/or the heavy chain or heavy chainfragment. These techniques may include steps of obtaining cloned genesfrom natural sources, or from synthetic oligonucleotide sequences, whichmay encode clostridial neurotoxin components including clostridialneurotoxin heavy chains, light chains or variants thereof, modifiedclostridial neurotoxin chains and/or fragments of the chains. Clonedgenes may also encode a targeting moiety.

The genes may be cloned into, for example, cloning vectors, such asphages or plasmids or phagemids. The recombinant vectors are transformedinto host cells, for example, into a prokaryotic cell, for example, E.coli. Proteins can be expressed and then isolated using conventionaltechniques.

Fusion genes may be used which encode more than one component of anagent. For example, a targeting moiety and a botulinum toxin heavy chainand/or light chain and/or a fragment of a heavy and/or a fragment of alight chain, can be produced from a single cloned gene as a fusionprotein. Alternatively, individual components obtained from recombinanttechniques can be chemically coupled to other components obtain fromother sources. For example, a synthetic or naturally occurring targetingmoiety component may be coupled to a recombinant L chain or to arecombinant fusion LH_(N). The linkages between the clostridialcomponents and the targeting moieties may include appropriate spacercomponents which may also be DNA encoded and included in the fusion geneconstruct.

In another embodiment, the required LH_(N), which may be a hybrid of anL chain and an H_(N) from different clostridial toxin types, isexpressed recombinantly as a fusion protein. Such LH_(N) hybrid may alsobe coupled to the targeting moiety, which may further include one ormore spacer regions between them.

In another embodiment of the invention the L chain of a clostridialneurotoxin, or a fragment of the L chain containing the endopeptidaseactivity, is expressed recombinantly as a fusion protein with the H_(N)of the H chain and the targeting moiety. The expressed fusion proteinmay also include one or more spacer regions. For example, the L chainmay be fused to H_(N) which is in turn fused to the targeting moiety. Inanother example, the H_(N) may be fused to the L chain which is in turnfused to the targeting moiety. Spacer components may be expressedrecombinantly between some or all of the components of an agent of theinvention.

In one embodiment of producing a hybrid of LH_(N), the L chain isobtained from botulinum toxin type B and the amine end segment of theH_(N) chain fragment is obtained from botulinum toxin type A. The H_(N)fragment of the botulinum toxin type A is produced according to themethod described by Shone C. C., Hambleton, P., and Melling, J. (1987,Eur. J. Biochem. 167,175-180) and the L chain of botulinum toxin type Baccording to the method of Sathyamoorthy, V. and DasGupta, B. R. (1985,J. Biol. Chem. 260, 10461-10466). The free cysteine on the amine endsegment of the H chain fragment of botulinum toxin type A is thenderivatized by the addition of a ten-fold molar excess of dipyridyldisulphide followed by incubation at 4 degrees C. overnight. The excessdipvridyl disulphide and the thiopyridone by product are then removed bydesalting the protein over a PD10 column (Pharmacia) into PBS.

The derivatized H_(N) is then concentrated to a protein concentration inexcess of 1 mg/ml before being mixed with an equimolar portion of Lchain from botulinum toxin type B (>1 mg/ml in PBS). After overnightincubation at room temperature the mixture is separated by sizeexclusion chromatography over Superose 6 (Pharmacia), and the fractionsanalyzed by SDS-PAGE. The chimeric LH_(N) is then available to produce aconjugated agent which includes a targeting moiety component.

The example described above is purely illustrative of the invention. Insynthesizing the agents, the coupling of the targeting moieties to theclostridial components, for example the modified clostridial neurotoxinsor fragments thereof, may be achieved via chemical coupling usingreagents and techniques known to those skilled in the art. Thus, anycoupling chemistry capable of covalently attaching the targetingmoieties of the agents to clostridial neurotoxin components and known tothose skilled in the art is covered by the scope of this application.

In one embodiment of the invention, there are provided methods for thetreatment of pain which comprise locally administering directly to aregion of inflammation and/or inflammation pain in a patient, intherapeutically effective doses, an agent of the invention. In anotherembodiment, the invention provides for administration of an agent of theinvention near the site of inflammation and/or inflammation pain in apatient, for example, within about 0.1 to about 100 cm, or for example,about 1 cm to about 10 cm from the site of inflammation and/orinflammation pain.

Known local drug administration methods suitable for administration maybe used including injection, with or without the use of a needle, and byinsertion of a controlled release implant. Routes of administrationinclude, without limitation, transdermal, peritoneal, subcutaneous,intramuscular, and intrarectal injection.

In another embodiment, a therapeutically effective dose of an agent ofthe invention is administered by systemic administration, for example,oral administration, intravenous administration or administration as aninhalant.

An agent, such as botulinum toxin component-substance P or componentsthat are substantially similar to substance P can require, according tothe methods of the present invention, from about 1 to 7 days to begin toachieve an effect upon a site of inflammation pain.

The amount of the agents administered can vary widely according to theparticular disorder being treated, its severity and other variouspatient variables including size, weight, age, and responsiveness totherapy.

Methods of the present invention provide for pain relief ranging fromabout 5% to about 100% in effectiveness. For example, pain may bereduced by about 20%, about 40%, about 50%, about 60%, about 80% orabout 100%.

Methods for determining the appropriate route of administration anddosage are generally determined on a case-by-case basis by the attendingphysician. Such determinations are routine to one of ordinary skill inthe art (see for example, Harrison's Principles of Internal Medicine(1998), edited by Anthony Fauci et al., 14^(th) edition, published byMcGraw Hill).

Generally, the dose of an agent to be administered will vary with theage, presenting condition and weight of the patient to be treated. Thepotency of the agent will also be considered. Agent (e.g. conjugate)potency is expressed as a multiple of the LD₅₀ value of an agent of theinvention for a mouse. A “U” or “unit” of an agent can be defined as theamount of toxin that kills 50% of a group of mice that were disease-freeprior to inoculation with the agent. Alternatively, potency may beexpressed as the LD₅₀ value of an agent that would be produced by anequal molar amount of botulinum toxin with a functional H_(C).

Agents of the invention can be administered in a dose of about 0.01units up to about 1,000 units. In one embodiment, individual dosages ofabout 1 unit to about 30 units are used. In another embodiment,individual dosages of about 30 units to about 60 units are used. Instill another embodiment, individual dosages of about 60 units to about180 units are used. Generally, the agents may be administered as acomposition at a dosage that is proportionally equivalent to, forexample, about 2.5 cc/100 units. Those of ordinary skill in the art willknow, or can readily ascertain, how to adjust these dosages for an agentof greater or lesser potency.

Preferably, the lowest therapeutically effective dosage will beadministered to the patient. The lowest therapeutic dosage is thatdosage which results in detection by the patient of a reduction in theoccurrence and/or magnitude of pain experienced by the patient, forexample, pain experience by a patient which is associated withneurogenic inflammation.

Methods for assessing or quantifying the amount of pain experienced by apatient are well known to those skilled in the art. For example, apatient can be given a pain assessment test in which the patientquantifies the degree of pain based on a scale. One example would beassigning the patient's pain a number based on a scale of 1 to 10, wherea “10” would indicate the worst degree of pain the patient mightimagine. A pain measure of 4 from an original pain score of 8 would be a50% reduction in pain. Thus, the amount of conjugate required to achievethat 50% reduction in pain could be considered 1 U of the clostridialtoxin component-targeting moiety conjugate. Alternatively, the patient'spain may be measured as the duration of pain. One unit of the conjugateof the invention would accordingly reduce the duration of pain by 50%.In addition, a number of physiological measures, such as heart rate,respiratory rate, blood pressure, and diaphoresis, may be used alone ortogether with the methods described above, to quantify the amount of thepatient's pain.

In an initial treatment, a low dosage may be administered at one site todetermine the patient's sensitivity to, and tolerance of, the agent.Additional administration of the same or different dosages may beperformed as necessary.

In one embodiment, an agent of the invention is administeredintramuscularly. However, for some indications, extramuscular injectionmay be the most efficacious route of administration. Such injection may,for example, be made subcutaneously or, preferably, perivascularly (toproduce infiltration of the agent into innervated tissue). In oneembodiment, the site for injection of the agent is in or near theextramuscular regions.

The agents may be administered by, for example, injection using a needleor by needleless injection. The injections may be repeated as necessary.

In needleless injection delivery methods, microprojectile drug particlesmay be coated with an agent and then discharged into the skin from anexternal delivery device. Depending on the discharge velocity and thedistance from the injection site, the drug particles penetrate throughthe stratum corneum to different layers of the epidermis, dermis andunderlying muscle.- As the microprojectiles penetrate through epidermaland dermal cells, or are deposited in these cells, the agent isreleased. Individual layers of skin cells or underlying muscle cells maybe targeted for the microprojectiles.

A range for administration of an agent, such as a LH_(N)-substance Pconjugate (or targeting moiety substantially similar to substance Pincluding substance P precursors, fragments and analogs), so as toachieve an antinociceptive effect in the patient treated may be fromabout 10⁻² U/kg to about 100 U/kg, for example between about 10⁻¹ U/kgto about 10 U/kg. Dosage for a particular agent may depend on factorssuch as the condition to be treated and the method of administration. Anappropriate dosage in a given circumstance may readily be determined bya physician of ordinary skill.

A range for administration of an agent, such as an a L chain-substance Pconjugate, (or targeting moiety substantially similar to substance Pincluding substance P precursors, fragments and analogs) so as toachieve an antinociceptive effect in the patient treated may be fromabout 10⁻² U/kg to about 100 U/kg, for example between about 10⁻¹ U/kgto about 10 U/kg. Dosage for a particular agent may depend on factorssuch as the condition to be treated and the method of administration. Anappropriate dosage in a given circumstance may readily be determined bya physician of ordinary skill.

Dosage for a particular agent may depend on factors such as thecondition to be treated and the method of administration. A physician ofordinary skill may readily determine an appropriate dosage in a givencircumstance.

The invention having been fully described, examples illustrating itspractice are set forth below. These examples should not, however, beconsidered to limit the scope of the invention, which is defined by theappended claims.

EXAMPLES Example 1 Methods for Determining Potency of Botulinum ToxinComponent-Targeting Moiety Conjugates

The traditional unit of measure for botulinum toxin potency is the mouseLD₅₀ unit. That is, one unit (1 U) of botulinum toxin is the amount thatkills 50% of a group of 18-20 gram female Swiss-Webster mice.

The unit of measure for potency of a botulinum toxin component-targetingmoiety conjugate may also be determined by LD₅₀ assays. In particular, 1U of the botulinum toxin component-targeting moiety component conjugate(for example, a LH_(N)-substance P conjugate) is the amount of theconjugate that kills 50% of a group of 18-20 gram female Swiss-Webstermice.

Alternatively, potency of botulinum toxin component-targeting moietycomponent conjugate may be determined by the amount of pain reduction ina patient induced by a measured amount of conjugate. For example, thepain reduction in a patient may be estimated to be 50% upon injection ofa measured amount of a conjugate into a site of inflammation. Thus, thepotency can be measured as the amount of conjugate that reduces apatient's pain by 50%.

Methods for assessing or quantifying the amount of pain experienced by asubject are well known to those skilled in the art. For example, asubject can be given a pain assessment test in which the subjectquantifies the degree of pain based on a scale. One example would beassigning the subject's pain a number based on a scale of 1 to 10, wherea “10” would indicate the worst degree of pain the subject mightimagine. A pain measure of 4 from an original pain score of 8 would be a50%-reduction in pain. Thus, the amount of conjugate required to achievethat 50% reduction in pain could be considered 1 U of the botulinumtoxin component-targeting moiety component conjugate. Alternatively, thesubject's pain may be measured as the duration of pain. One unit of theconjugate of the invention would accordingly reduce the duration of painby 50%. In addition, a number of physiological measures, such as heartrate, respiratory rate, blood pressure, and diaphoresis, may be usedindividually or in combination to quantify a subject's pain. Theseprocedures may also be used in combination with the subjective methodsdescribed above, to quantify the amount of the subject's pain.

Example 2 Treatment of Neurogenic Inflammation Pain

A patient, age 45, experiencing acute neurogenic inflammation pain istreated by intravenous administration with between about 0.1 U/kg andabout 30 U/kg, (for example, about 4 U), of an agent comprising anLH_(N) botulinum toxin type A-substance P conjugate. Within 1-7 daysafter agent administration the patient's pain is substantiallyalleviated. The duration of pain reduction is from about 2 to about 6months.

Example 3 Treatment of Neurogenic Inflammation Pain

A patient, age 36, experiencing inflammation pain of neurogenic originis treated by direct administration to the site of pain of between about0.1 U/kg and about 30 U/kg, (for example, from about 1 U to about 10 U),of an agent comprising a botulinum toxin type. A L chain-substance Pconjugate. A physician of ordinary skill may readily determine thedosage, site of injection, and frequency of administration. Within 1 -7days the pain symptoms are substantially alleviated. The duration ofpain reduction is from about 2 to about 6 months.

Example 4 Treatment of Neurogenic Inflammation Pain

A patient, age 45, experiencing acute neurogenic inflammatory pain istreated by administration, for example by injection directly to a siteof pain with an amount of an agent comprising botulinum toxin type A, B,C, D, E, F and/or G H_(N) and type A, B, C, D, E, F and/or G Lchain-substance P, substance P analog and/or substance P fragmentconjugate that reduces pain in the subject by about 50%. A physician ofordinary skill may readily determine the dosage, method ofadministration, and frequency of administration. Within 1-7 days afteragent administration the patient's pain is alleviated by about 50% (inparticular, the patient's pain score is originally a 6, and aftertreatment, the patient scores his pain as a 3). The duration of painreduction is from about 2 to about 6 months.

Example 5 Treatment of Neurogenic Inflammation Pain

A patient, age 36, experiencing neurogenic inflammation pain (score of8) is treated by intravenous administration of about 1 U to about 20 Uof an agent comprising a botulinum toxin type A, B, C, D, E, F and/or GL chain -substance P, substance P analog and/or substance P fragmentconjugate that reduces the pain in the subject by about 80% (score ofabout 1 to about 2). A physician of ordinary skill may readily determinethe dosage and frequency of administration. Within 1-7 days the painsymptoms are reduced by about 80%. The duration of pain reduction isfrom about 2 to about 6 months.

Example 6 Treatment of Fibromyalgia

A 37-year old woman complains of pain “all over,” specifically in theocciput, neck, shoulders, lower back, hips, and right leg.

At the time of evaluation, the patient complains specifically ofoccipital headache, which she describes as burning and aching. Shecomplains of neck pain and stiffness. Aching pain in the upper back andshoulders is constant, as is pain in a band-like fashion across thelower back. She also notes a great increase in her upper back and neckpain. The patient injures her lower back one year prior to thisevaluation-while working as a nurse's aide as she transfers a patient.She subsequently continues to experience lower back, hip, and right legpain and stiffness.

Upon examination, it is found that the patient tests positive for painsensitivity in 11 of the 18 fibromyalgia tender points. Specifically,tenderness is present in the left and-right occiput, the left and rightcervical regions, the left and right trapizious, the left and rightgluteal, the left and right supraspinatus and the right greatertrochanter. A diagnosis of fibromyalgia is made.

The patient is treated by injection of about 1 to about 40 units ofbotulinum toxin type A LH_(N)-substance P conjugate into tendoninsertion sites near the perceived sources of pain. Within one week oftreatment, the patient notes a substantial decrease in pain feltthroughout her body. Pain relief lasts for approximately eight months.

Example 7 Treatment of Myofascial Pain Syndrome

A physical examination reveals a 38-year old woman who suffers nervedamage in an auto accident at the age of 28. She suffers from a steadypain in addition to allodynia in her legs. The patient walks with anormal gait and is able to perform heel walk and toe walk withoutdifficulty. Range of motion of the lumbar and cervical spine iscomplete. Motor and sensory examination of the upper and lowerextremities reveals mild S1 sensory loss on the right side. Reflexes areintact at the knees and ankles.

Radiographics of the cervical and lumbar spine are essentially normaldemonstrating only mild degenerative changes.

The patient is diagnosed with myofacial pain syndrome and is treatedwith injections of about 1 to about 60 units of botulinum toxin type A Lchain, type B H_(N) hybrid LH_(N)-substance P conjugate. A physician ofordinary skill may readily determine the dosage, method ofadministration, and frequency of administration. After approximately 6days the patient reports herself to be pain free. The relief from painlasts approximately 6 months at which time the injections are repeated.

Example 8 Treatment of Irritable Bowel

A 30 year old man visits his family physician with chief complaints ofpersistent pain in the upper and lower back, indigestion, constanttiredness and frequent urination.

The man discloses bouts of gastrointestinal discomfort, both before andafter meals. The patient denies any drug use or cigarette smoking,except for an occasional alcoholic drink. Visual observation reveals aman not under distress. Cranial tests are normal. Reflexes are normal. Ablood chemistry is ordered. With an exception of a slightly lower K+level, all other values are normal. Antinuclear antibody, rheumatoidfactor, creatine kinase, magnesium, lyme titre, and thyroid functiontests are negative. A diagnosis of irritable bowel syndrome is made. Thephysician prescribes injections of about 1 to about 10 units ofbotulinum toxin type A LH_(N)-substance P conjugate into the wall of thelower small intestine. After one week, the patient reports only mildrelief. A further injection is made using about 1 to about 40 units ofthe same conjugate agent. Pain relief of up to 80% is realized one weekafter this treatment.

Example 9 Treatment of Migraine Headache

A 34-year-old woman seeks medical attention for migraine headache. Thepatient reports the symptoms persisting for approximately 5 years. Inaddition, the patient reports an inability to obtain restful sleep. Thepatient has been prescribed numerous medications over the yearsincluding tylenol 111, Propanolol hydrochloride, Dihydroergotaminemesylate, Naratriptan hydrochloride, Sumatriptan succinate andZolmitriptan. All have met with moderate to little effect.

The patient is intravenously injected with about 1 to about 120 units ofa botulinum toxin type A L chain, type B H_(N), LH_(N)-substance Pconjugate conjugate.

At 1 week a physical examination reveals no sign of migraine headachepain. Pain relief last for approximately 9 months.

Example 10 Treatment of Arthritis

A patient, age 45, complains of pain in the back and left hip. Thepatient also reports symptoms of pain and lack of mobility in his leftwrist, and in several of his fingers. The patient is diagnosed withrheumatoid arthritis. The patient is treated by an injection of about 1to about 50 units of a botulinum toxin type A L chain, type B H_(N)hybrid LH_(N)-substance P conjugate and or a botulinum toxin type A Lchain -substance P conjugate into or near the regions of pain. Aphysician of ordinary skill may readily determine the specific dosage,site of injection, and frequency of administration. Within 1-7 daysafter administration the patient's pain is substantially alleviated. Theduration of the pain alleviation is from about 1 to about 6 months.

Example 11 Treatment of Interstitial Cystitis

Upon examination, a 56 year old woman complains of chronic pelvic pain,pain with sexual relations, sleep difficulties and incontinence. Thepatient also reports symptoms of irritable bowel and fatigue. She statesthat she is suffering from these symptoms for over a year and that justrecently the pain is increasing substantially. A diagnosis ofinterstitial cystitis is made. The patient is treated by injection ofabout 2 to about 200 units of botulinum toxin type A LH_(N)-substance Pconjugate into the bladder wall. A physician of ordinary skill mayreadily determine the specific dosage, site of injection, and frequencyof administration. Within 1-7 days after modified neurotoxinadministration the patient's pain is substantially alleviated. Theduration of the pain alleviation is from about 7 to about 27 months.

Example 12 Treatment of Pain Associated With Fibromyalgia Tender Points

A 36 year old woman has a 15 year history of chronic pain in the uppertorso area. Fifteen years prior to evaluation she notes a decrease inmobility in her left elbow and knees. The pain in the left side of herbody is thought to be worse than in the right. Upon examination it isrevealed that the patient is pain sensitive to firm pressure that isapplied sequentially to eight of the eighteen fibromyalgia tenderpoints. In order for a diagnosis of fibromyalgia to be made, 11 of the18 fibromyalgia tender points must be pain sensitive to application ofpressure. Since this standard is not met, the patient is not diagnosedas having fibromyalgia. She is treated with antidepressant medicationincluding amytriptyline (Elavil). The medication has little effect onthe level of pain experienced by the patient.

The patient is injected with about 1 to about 20 units of botulinumtoxin type A L chain-substance P conjugate into the sites where thepatient perceives the pain to originate. A physician of ordinary skillmay readily determine the specific dosage, site of injection, andfrequency of administration.

Several days after the injections she notes substantial improvement inher pain. This gradually improves over a 2 to 3 week period in which shenotes increased mobility in her elbow and knee joints. The patientstates that the pain is better than at any time in the last 4 years. Theimproved condition persists for up to 6 months.

1. A method for treating migraine headache pain, the method comprisingthe step of administering a therapeutically effective amount of about0.01 units to about 1,000 units of an agent, the agent comprising abotulinum toxin light chain, a botulinum toxin heavy chain H_(N) portionand a substance P component effective in binding to a substance Preceptor.
 2. The method according to claim 1, wherein the botulinumtoxin light chain is selected from the group consisting of a botulinumtoxin serotype A light chain, a botulinum toxin serotype B light chain,a botulinum toxin serotype C1 light chain, a botulinum toxin serotype Dlight chain, a botulinum toxin serotype E light chain, a botulinum toxinserotype F light chain and a botulinum toxin serotype G light chain. 3.The method according to claim 1, wherein the botulinum toxin heavy chainH_(N) portion is selected from the group consisting of a botulinum toxinserotype A heavy chain H_(N) portion, a botulinum toxin serotype B heavychain H_(N) portion, a botulinum toxin serotype C1 heavy chain H_(N)portion, a botulinum toxin serotype D heavy chain H_(N) portion, abotulinum toxin serotype E heavy chain H_(N) portion, a botulinum toxinserotype F heavy chain H_(N) portion and a botulinum toxin serotype Gheavy chain H_(N) portion.
 4. The method according to claim 1, whereinthe botulinum toxin light chain comprises a botulinum toxin serotype Alight chain.
 5. The method according to claim 1, wherein the botulinumtoxin heavy chain H_(N) portion comprises a botulinum toxin serotype Aheavy chain H_(N) portion.
 6. The method according to claim 1, whereinthe substance P component comprises substance P.
 7. The method accordingto claim 1, wherein the agent is administered local to the site of pain.8. The method according to claim 1, wherein the agent is administeredsystemically.
 9. The method according to claim 1, wherein the agent isadministered subcutaneously.
 10. The method according to claim 1,wherein the agent is administered intramuscularly.
 11. The methodaccording to claim 1, wherein the amount of the agent administered isfrom about 0.01 U/kg to about 1 U/kg.
 12. The method according to claim1, wherein the amount of the agent administered is from about 0.1 U/kgto about 10 U/kg.
 13. The method according to claim 1, wherein theamount of the agent administered is from about 1 U/kg to about 100 U/kg.14. The method according to claim 1, wherein the amount of the agentadministered is from about 1 U/kg to about 200 U/kg.